草莓FaWRKY31的克隆、亚细胞定位及表达特性分析

Cloning,Subcellular Location and Expression Analysis of FaWRKY31 in Fragaria × ananassa

为明确草莓FaWRKY31的序列特征、表达特性和亚细胞定位情况,通过同源克隆的方法从八倍体草莓‘红颜’中得到3条长度为1644 bp、1条长度为1669 bp的cDNA序列和两条长度约为2000 bp的启动子序列。生物信息学分析表明:长度为1669 bp的FaWRKY31-like因插入了一段25 bp的短序列导致开放阅读框提前终止,缺失了WRKY结构域和C2H2锌指结构。启动子的序列分析结果表明:FaWRKY31与森林草莓FvWRKY31的启动子序列存在较大差异,相似性仅为70%,FaWRKY31的启动子序列发生了多个片段的缺失和插入,可能导致其与森林草莓FvWRKY31有不同的诱导特性。qRT-PCR结果表明:FaWRKY31在草莓的根、茎、叶、花及果实中均有表达,根中的表达量最高,全红期果实中的最低。FaWRKY31能同时响应红光和蓝光的调控,抑制其在草莓果实中的表达。此外,FaWRKY31能在不同程度上响应低钾、低磷、低温、ABA、盐害、干旱这6种非生物胁迫,且低温、ABA及干旱都显著抑制FaWRKY31的表达。亚细胞定位结果显示FaWRKY31蛋白定位于细胞核内。

To clarify the sequence characteristics,expression patterns and subcellular localization of FaWRKY31 in octoploid strawberry cultivar‘Benihoppe’,three CDS sequences with a length of 1644 bp,one CDS sequence with a length of 1669 bp and two promoter sequences about 2000 bp were isolated by homology cloning strategy.Bioinformatics analysis indicated that the open reading frame of FaWRKY31-like with a length 1669 bp was terminated prematurely because of a 25 bp sequence insertion,resulting the truncation of WRKY domain as well as C2H2 Zinc finger.Sequence analysis of promoter showed that there was a large divergence between FaWRKY31 promoter sequence and FvWRKY31.The similarity of those two promoter sequences was 70%,FaWRKY31 promoter existed some deletion and insertion of multiple fragments,which may lead to different inducing characteristics compared with FvWRKY31(F.vesca).The gene expression level of FaWRKY31 evaluated by q-PCR showed that FaWRKY31 was detected in all analyzed tissues.The highest relative expression level was in roots,followed by stems and functional leaves and the lowest relative expression was in fruits.FaWRKY31 could be induced by red and blue light treatments,and be downregulated by blue and red light in strawberry fruits.FaWRKY31 can also significantly induced by low potassium,low phosphorus,ABA,drought,salt stress and low temperature in different degrees,and low temperature,ABA,drought had negative effects on FaWRKY31.Subcellular location of FaWRKY31 showed that FaWRKY31 was a nuclear locatized protein.